Recombinant MVA virus, and the use thereof

ABSTRACT

The present invention relates to recombinant vaccinia viruses derived from the modified vaccinia virus Ankara (MVA) and containing and capable of expressing foreign genes which are inserted at the site of a naturally occurring deletion in the MVA genome, and the use of such recombinant MVA viruses for the production of polypeptides, e.g. antigens or therapeutic agents, or viral vectors for gene therapy, and the use of such recombinant MVA viruses encoding antigens as vaccines.

RELATED APPLICATION(S)

This application is a division of U.S. application Ser. No. 10/147,284,filed May 15, 2002, which is a continuation of U.S. application Ser. No.09/002,443, filed Jan. 2, 1998, which is a continuation of InternationalApplication No. PCT/EP96/02926, which designated the United States andwas filed on Jul. 3, 1996, published in English, which claims thebenefit of Danish Patent Application No. DK 0782/95, filed Jul. 4, 1995.The entire teachings of the above application(s) are incorporated hereinby reference

BACKGROUND OF THE INVENTION

Vaccinia virus, a member of the genus Orthopoxvirus in the family ofPoxviridae, was used as live vaccine to immunize against the humansmallpox disease. Successful world-wide vaccination with vaccinia virusculminated in the eradication of variola virus, the causative agent ofthe smallpox (The global eradication of smallpox. Final report of theglobal commission for the certification of smallpox eradication. Historyof Public Health, No. 4, Geneva: World Health Organization, 1980). Sincethat WHO declaration, vaccination has been universally discontinuedexcept for people at high risk of poxvirus infections (e.g. laboratoryworkers).

More recently, vaccinia viruses have also been used to engineer viralvectors for recombinant gene expression and for the potential use asrecombinant live vaccines (Mackett, M. et al., P.N.A.S. USA,79:7415-7419 (1982); Smith, G. L et al., Biotech. and GeneticEngineering Reviews 2:383-407, (1984)). This entails DNA sequences(genes) which code for foreign antigens being introduced, with the aidof DNA recombination techniques, into the genome of the vacciniaviruses. If the gene is integrated at a site in the viral DNA which isnon-essential for the life cycle of the virus, it is possible for thenewly produced recombinant vaccinia virus to be infectious, that is tosay able to infect foreign cells and thus to express the integrated DNAsequence (EP Patent Applications No. 83,286 and No. 110,385)). Therecombinant vaccinia viruses prepared in this way can be used, on theone hand, as live vaccines for the prophylaxis of infectious diseases,on the other hand, for the preparation of heterologous proteins ineukaryotic cells.

Recombinant vaccinia virus expressing the bacteriophage T7 RNApolymerase gene allowed the establishment of widely applicableexpression systems for the synthesis of recombinant proteins inmammalian cells (Moss, B., et al., Nature, 348:91-92 (1990)). In allprotocols, recombinant gene expression relies on the synthesis of the T7RNA polymerase in the cytoplasm of eukaryotic cells. Most popular becamea protocol for transient-expression (Fuerst, T. R., et al., Proc. Natl.Acad. Sci. USA, 83:8122-8126 (1986) and U.S. patent application Ser. No.7,648,971)). First, a foreign gene of interest is inserted into aplasmid under the control of the T7 RNA polymerase promoter. In thefollowing, this plasmid is introduced into the cytoplasm of cellsinfected with a recombinant vaccinia virus producing T7 RNA polymeraseusing standard transfection procedures.

This transfection protocol is simple because no new recombinant virusesneed to be made and very efficient with greater than 80% of the cellsexpressing the gene of interest (Elroy-Stein, O. and Moss, B., Proc.Natl. Acad. Sci. USA, 87:6743-6747 (1990)). The advantage of thevaccinia virus/T7 RNA polymerase hybrid system over other transientexpression systems is very likely its independence on the transport ofplasmids to the cellular nucleus. In the past, the system has beenextremely useful for analytical purposes in virology and cell biology(Buonocore, L. and Rose, J. K, Nature, 345:625-628, (1990); Pattnaik, A.K and Wertz, G. W., Proc. Natl. Acad. Sci. USA, 88:1379-1383 (1991);Karschin, A. et al., FEBS Lett. 278: 229-233 (1991), Ho, B.Y. et al.,FEBSLett., 301:303-306 (1992); Buchholz, C. J. et al., Virology,204:770-776 (1994)). However, important future applications of thevaccinia virus/T7 RNA polymerase hybrid system, as e.g. to generaterecombinant proteins or recombinant viral particles for noveltherapeutic or prophylactic approaches in humans, might be hindered bythe productive replication of the recombinant vaccinia vector.

Vaccinia virus is infectious for humans and upon vaccination during thesmallpox eradication campaign occasional serious complications wereobserved. The best overview about the incidence of complications isgiven by a national survey in the United States monitoring vaccinationof about 12 million people with a vaccine based on the New York CityBoard of Health strain of vaccinia virus (Lane, J. et al. New Engl. J.Med., 281:1201-1208, (1969)). Therefore the most exciting possibility touse vaccinia virus as vector for the development of recombinant livevaccines has been affected by safety concerns and regulations.Furthermore, most of the recombinant vaccinia viruses described in theliterature are based on the Western Reserve strain of vaccinia virus. Onthe other hand, it is known that this strain has a high neurovirulenceand is thus poorly suited for use in humans and animals (Morita et al.,Vaccine, 5:65-70 (1987)).

For vector applications health risks would be lessened by the use of ahighly attenuated vaccinia virus strain. Several such strains ofvaccinia virus were especially developed to avoid undesired side effectsof smallpox vaccination. Thus, the modified vaccinia virus Ankara (MVA)has been generated by long-term serial passages of the Ankara strain ofvaccinia virus (CVA) on chicken embryo fibroblasts (for review see Mayr,A., et al., Infection, 3:6-14 (1975); Swiss Patent No. 568,392)). TheMVA virus was deposited in compliance with the requirements of theBudapest Treaty at CNCM (Institut Pasteur, Collection Nationale deCultures Microorganisms, 25, rue du Docteur Roux, 75724 Paris Cedex 15)on Dec. 15, 1987 under Depositary No. I-721. MVA is distinguished by itsgreat attenuation, that is to say by diminished virulence orinfectiosity while maintaining good immunogenicity. The MVA virus hasbeen analyzed to determine alterations in the genome relative to thewild CVA strain. Six major deletions of genomic DNA (deletion I, II,III, IV, V, and VI) totaling 31,000 base pairs have been identified(Meyer, H., et al., J. Gen. Virol. 72:1031-1038 (1991)). The resultingMVA virus became severely host cell restricted to avian cells.

Furthermore, MVA is characterized by its extreme attenuation. Whentested in a variety of animal models, MVA was proven to be avirulenteven in immunosuppressed animals. More importantly, the excellentproperties of the MVA strain have been demonstrated in extensiveclinical trials (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167:375-390(1987), Stickl et al., Dtsch. med. Wschr. 99:2386-2392 (1974)). Duringthese studies in over 120,000 humans, including high risk patients, noside effects were associated with the use of MVA vaccine.

MVA replication in human cells was found to be blocked late in infectionpreventing the assembly to mature infectious virions. Nevertheless, MVAwas able to express viral and recombinant genes at high levels even innon-permissive cells and was proposed to serve as an efficient andexceptionally safe gene expression vector (Sutter, G. and Moss, B.,Proc. Natl. Acad. Sci. USA 89:10847-10851 (1992)). Recently, novelvaccinia vector systems were established on the basis of MVA, havingforeign DNA sequences inserted at the site of deletion III within theMVA genome or within the TK gene (Sutter, G. and Moss, B. Dev. Biol.Stand. Basel, Karger 84:195-200 (1995) and U.S. Pat. No. 5,185,146)).

To further exploit the use of MVA a novel possible way to introduceforeign genes by DNA recombination into the MVA strain of vaccinia virushas been sought. Since the intention was not to alter the genome of theMVA virus, it was necessary to use a method which complied with thisrequirement. According to the present invention a foreign DNA sequencewas recombined into the viral DNA precisely at the site of a naturallyoccurring deletion in the MVA genome.

SUMMARY OF THE INVENTION

The present invention thus, inter alia, comprises the following, aloneor in combination:

-   A recombinant MVA virus containing and capable of expressing at    least one foreign gene inserted at the site of a naturally occurring    deletion within the MVA genome;-   a recombinant MVA virus as above containing and capable of    expressing at least one foreign gene inserted at the site of    deletion II within the MVA genome;-   a recombinant MVA virus as above wherein the foreign gene codes for    a marker, a therapeutic gene or an antigenic determinant;-   a recombinant MVA virus as above wherein the foreign gene codes for    an antigenic determinant from a pathogenic virus, a bacteria, or    other microorganism, or from a parasite, or a tumor cell;-   a recombinant MVA virus as above wherein the foreign gene codes for    an antigenic determinant from Plasmodium Falciparum, Mycobacteria,    Herpes virus, influenza virus, hepatitis, or human immunodeficiency    viruses.-   a recombinant MVA virus as above wherein the antigenic determinant    is HIV nef or human tyrosinase;-   a recombinant MVA virus as above which is MVA-LAInef or MVA-hTYR;-   a recombinant MVA virus as above wherein the foreign gene codes for    T7 RNA polymerase;-   a recombinant MVA virus as above which is MVA-T7 pol;-   a recombinant MVA virus as above wherein the foreign gene is under    transcriptional control of the vaccinia virus early/late promoter    P7.5;-   recombinant MVA viruses as above essentially free from viruses being    able to replicate in human cells;-   the use of a recombinant MVA virus as above for the transcription of    DNA sequences under transcriptional control of a T7 RNA polymerase    promoter;-   a eukaryotic cell infected by a recombinant MVA virus as any above;-   a cell infected by a recombinant MVA virus as above wherein the    foreign gene code for T7 RNA polymerase;-   a cell infected by a recombinant MVA virus as above wherein the    foreign gene code for T7 RNA polymerase, additionally containing one    or more expression vectors carrying one or more foreign genes under    transcriptional control of a T7 RNA polymerase promoter;-   the use of cells as above for the production of the polypeptides    encoded by said foreign genes comprising:    -   a) culturing said cells under suitable conditions, and    -   b) isolating the polypeptides encoded by said foreign genes.-   a cell infected by a recombinant MVA virus as above wherein the    foreign gene code for T7 RNA polymerase, additionally containing    expression vectors carrying viral genes, and/or a viral vector    construct encoding the genome of a viral vector under    transcriptional control of a T7 RNA polymerase promoter;-   the use of a cells as above for the production viral particles    comprising:    -   a) culturing said cells under suitable conditions, and    -   b) isolating the viral particles;-   a cell infected by a recombinant MVA virus as above wherein the    foreign gene code for T7 RNA polymerase, additionally containing    -   a) an expression vector carrying a retroviral vector construct        capable of infecting and directing the expression in target        cells of one or more foreign genes carried by said retroviral        vector construct, and    -   b) one or more expression vectors carrying the genes encoding        the polypeptides required for the genome of said retroviral        vector construct to be packaged under transcriptional control of        a T7 RNA polymerase promoter; the use of cells as above for the        production of retroviral particles comprising    -   a) culturing said cells under suitable conditions, and    -   b) isolating the retroviral particles;-   a vaccine containing a recombinant MVA virus as above wherein the    foreign gene code for an antigenic determinant in a physiologically    acceptable carrier;-   the use of a recombinant MVA virus as above wherein the foreign gene    code for an antigenic determinant preparation of a vaccine;-   the use of a vaccine as above for the immunization of a living    animal body, including a human;-   the use of a vaccine as above containing MVA-LAInef for the    prevention or treatment of HIV infection or AIDS;-   the use of a vaccine as above containing MVA-hTYR for the prevention    or treatment of melanomas;-   a vaccine comprising as a first component, a recombinant MVA virus    as above wherein the foreign gene code for T7 RNA polymerase in a    physiologically acceptable carrier, and as a second component a DNA    sequence carrying an antigenic determinant under transcriptional    control of a T7 RNA polymerase promoter in a physiologically    acceptable carrier, the two components being contained together or    separate;-   the use of a vaccine as above for the immunization of a living    animal body, including a human, comprising inoculation of said    living animal body, including a human, with the first and second    component of the vaccine either simultaneously or with a timelag    using the same inoculation site; and-   The term “gene” means any DNA sequence which codes for a protein or    peptide.

The term “foreign gene” means a gene inserted in a DNA sequence in whichit is not normally found.

The foreign gene can be a marker gene, a therapeutic gene, a geneencoding an antigenic determinant, or a viral gene, for example. Suchgenes are well known in the art.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic map of the genome of MVA and plasmid for insertionof foreign DNA by homologous recombination: HindIII restriction siteswithin the genome of MVA are indicated at the top; the 900-bpHindIII-HindIII N fragment that overlaps the junction of deletion IIwithin the MVA genome is shown; MVA DNA sequences adjacent to deletionII (flank 1 and flank 2) were amplified by PCR and used for theconstruction of insertion plasmid pUC II LZ.

FIG. 2 is a schematic map of pUC II LZ P7.5: MVA vector plasmid forinsertion into deletion II containing P11-LacZ expression cassette andthe vaccinia virus early/late promoter P7.5 to express genes of interestthat can be cloned into the SmaI site of the plasmid.

FIG. 3 is a schematic map of pUCII LZdel P7.5: MVA vector plasmid forinsertion of foreign genes at the site of deletion II in the MVA genome,containing a self-deleting P11-LacZ expression cassette and the vacciniavirus early/late promoter P7.5 to express genes of interest that can becloned into the SmaI/Notl cloning site of the plasmid.

FIG. 4 is a schematic map of the construction of recombinant virusMVA-T7pol: schematic maps of the MVA genome (HindIII restrictionendonuclease sites) and the vector plasmid pUC II LZ T7pol that allowsinsertion of the T7 RNA polymerase gene at the site of deletion IIwithin the HindIII N fragment of the MVA genome.

FIG. 5 is a schematic map of the construction of MVA-LAInef: schematicmaps of the MVA genome (HindIII restriction endonuclease sites) and thevector plasmid pUC II LZdel P7.5-LAInef that allows insertion of the nefgene of HIV-1 LAI at the site of deletion II within the HindIII Nfragment of the MVA genome.

FIG. 6 is a schematic map of the construction of MVA-hTYR: schematicmaps of the MVA genome (HindIII restriction endonuclease sites) and thevector plasmid pUC II LZdel P7.5-TYR that allows insertion of the humantyrosinase gene at the site of deletion II within the HindIII N fragmentof the MVA genome.

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide a recombinant MVAvirus which can serve as an efficient and exceptionally safe expressionvector.

Another object of the present invention is to provide a simple,efficient and safe method for the production of polypeptides, e.g.antigens or therapeutic agents, recombinant viruses for vaccines andviral vectors for gene therapy.

Still another object of the present invention is to provide anexpression system based on a recombinant MVA virus expressing T7 RNApolymerase, and methods for the production of polypeptides, e.g.antigens or therapeutic agents, or for generating viral vectors for genetherapy or vaccines, based on this expression system.

The Present Invention

Modified vaccinia virus Ankara (MVA), a host range restricted and highlyattenuated vaccinia virus strain, is unable to multiply in human andmost other mammalian cell lines tested. But since viral gene expressionis unimpaired in non-permissive cells the recombinant MVA virusesaccording to the invention may be used as exceptionally safe andefficient expression vectors.

Recombinant MVA Viruses Encoding an Antigenic Determinant

In one embodiment, the present invention relates to recombinant MVAvaccinia viruses which contain a gene which codes for a foreign antigen,preferably of a pathogenic agent, and vaccines containing such a virusin a physiologically acceptable form. The invention also relates tomethods for the preparation of such recombinant MVA vaccinia viruses orvaccines, and to the use of these vaccines for the prophylaxis ofinfections caused by such pathogenic agents.

In a preferred embodiment of the invention, the foreign gene inserted inthe MVA virus is a gene encoding HIV nef.

We have constructed recombinant MVA viruses that allow expression of theHIV-1 nef gene under the control of the vaccinia virus early/latepromoter P7.5. The regulatory Nef protein of primate lentiviruses issynthesized early in the viral replication cycle and has been shown tobe essential for high titer virus replication and disease induction invivo. This suggests that HIV Nef might play a crucial role in AIDSpathogenesis. The molecular mechanism(s) by which Nef contributes toincreased viral infectivity and to HIV pathogenicity need to be furtherelucidated. However, Nef is immunogenic and Nef-specific antigen can beused as a vaccine against HIV infection and AIDS.

In this context, the recombinant MVA virus expressing the HIV nef genecan be used for immunization of human beings, on one hand, as aprophylactic vaccine against human HIV, and on the other hand, forimmunotherapy of HIV infected or AIDS patients. Furthermore, therecombinant MVA virus expressing the HIV nef gene can be used for theproduction of recombinant HIV Nef protein.

In another preferred embodiment of the invention the foreign geneinserted in the MVA virus is a gene encoding human tyrosinase.

We have constructed recombinant MVA viruses that allow expression of thehuman tyrosinase gene under the control of the vaccinia virus early/latepromoter P7.5. Recently, human tyrosinase was identified as amelanoma-specific tumor antigen that allows generation of anti-tumorcytolytic T-lymphocytes (Beichard, V., et al., J. Exp. Med., 178:489-495(1993)). Since among normal cells, only melanocytes appear to expressthe tyrosinase gene, tyrosinase is a useful target antigen forimmunotherapy of melanomas. Therefore, the recombinant MVA virusexpressing the human tyrosinase gene can be used in melanoma patients toinduce immune responses that provoke tumor rejection or preventmetastasis. Recombinant MVA virus expressing the human tyrosinase genecan be used directly as an anti-melanoma vaccine, or the virus can beused to prepare anti-melanoma vaccines. In one example, the recombinantMVA virus expressing the human tyrosinase gene can be used for theproduction of recombinant tyrosinase protein which is used as antigen invaccine preparations. In another example, using the recombinant MVAvirus expressing the human tyrosinase gene as expression vector, cellsderived from a tumor patient can be modified in vitro to expresstyrosinase and then transferred back to the patient to induce anti-tumorimmune responses. A vaccine prepared on the basis of recombinant MVAexpressing the human tyrosinase gene can be used either parenterally orlocally at the site of the tumor. To prevent tumor metastasis or tophenotypically change the tumor e.g. in size, shape, consistency,vascularization or other features. A vaccine prepared on the basis ofrecombinant MVA expressing the human tyrosinase gene can be used before,during, or after surgical extirpation of the tumor.

For the preparation of vaccines, the MVA vaccinia viruses according tothe invention are converted into a physiologically acceptable form. Thiscan be done based on the experience in the preparation of MVA vaccinesused for vaccination against smallpox (as described by Stickl, H. etal., Dtsch. med. Wschr. 99:2386-2392 (1974)). Typically, about 10⁶-10⁸particles of the recombinant MVA are freeze-dried in 100 ml ofphosphate-buffered saline (PBS) in the presence of 2% peptone and 1%human albumin in an ampoule, preferably a glass ampoule. Thelyophilisate can contain extenders (such as mannitol, dextran, sugar,glycine, lactose or polyvinylpyrrolidone) or other aids (such asantioxidants, stabilizers, etc.) suitable for parenteral administration.The glass ampoule is then sealed and can be stored, preferably attemperatures below −20° C., for several months.

For vaccination or therapy the lyophilisate can be dissolved in 0.1 to0.5 ml of an aqueous solution, preferably physiological saline, andadministered either parenterally, for example by intramuscularinoculation or locally, for example by inoculation into a tumor or atthe site of a tumor. Vaccines or therapeutics according to the inventionare preferably injected intramuscularly (Mayr, A. et al., Zbl. Bakt.Hyg., I. Abt. Orig. B 167:375-390 (1978)). The mode of administration,the dose and the number of administrations can be optimized by thoseskilled in the art in a known manner. It is expedient where appropriateto administer the vaccine several times over a lengthy period in orderto obtain appropriate immune responses against the foreign antigen.

The Use of Recombinant MVA Viruses for the Production of HeterologousPolypeptides

The recombinant MVA vaccinia viruses according to the invention can alsobe used to prepare heterologous polypeptides in eukaryotic cells. Thisentails cells being infected with the recombinant vaccinia viruses. Thegene which codes for the foreign polypeptide is expressed in the cells,and the expressed heterologous polypeptide is isolated. The methods tobe used for the production of such heterologous polypeptides aregenerally known to those skilled in the art (EP-A-206,920 andEP-A-205,939). The polypeptides produced with the aid of the recombinantMVA viruses are, by reason of the special properties of the MVA viruses,more suitable for use as medicaments in humans and animals.

Recombinant MVA Viruses Encoding T7 RNA Polymerase and the Use Thereoffor the Expression of DNA Sequences Under Transcriptional Control of aT7 RNA Polymerase Promoter

In a further embodiment of the present invention we have constructedrecombinant MVA viruses that allow expression of the bacteriophage T7RNA polymerase gene under the control of the vaccinia virus early/latepromoter P7.5. The usefulness of MVA-T7pol recombinant viruses asexpression system has been tested in transient transfection assays toinduce expression of recombinant genes under the control of a T7 RNApolymerase promoter. Using the E. coli chloramphenicol acetyltransferase(CAT) gene as a reporter gene we found that MVA-T7pol induced CAT geneexpression as effectively as a vaccinia/T7pol recombinant virus derivedfrom the replication-competent WR strain of vaccinia virus.

The MVA/T7 polymerase hybrid system according to the invention can thusbe used as a simple, efficient and safe mammalian expression system forproduction of polypeptides in the absence of productive vaccinia virusreplication.

This expression system can also be used for generating recombinant viralparticles for vaccination or gene therapy by transformation of celllines infected with recombinant MVA expressing T7 RNA polymerase, withDNA-constructs containing all or some of the genes, and the genome orrecombinant genome necessary for generating viral particles, e.g MVAparticles or retroviral particles, under transcriptional control of a T7RNA polymerase promoter.

Retroviral vector systems consist of two components:

-   -   1) the retroviral vector itself is a modified retrovirus (vector        plasmid) in which the genes encoding for the viral proteins have        been replaced by therapeutic genes and marker genes to be        transferred to the target cell. Since the replacement of the        genes encoding for the viral proteins effectively cripples the        virus it must be rescued by the second component in the system        which provides the missing viral proteins to the modified        retrovirus.

The second component is:

-   -   2) a cell line that produces large quantities of the viral        proteins, however lacks the ability to produce replication        competent virus. This cell line is known as the packaging cell        line and consists of a cell line transfected with one or more        plasmids carrying the genes (genes encoding the gag, pol and env        polypeptides) enabling the modified retroviral vector to be        packaged.

To generate the packaged vector, the vector plasmid is transfected intothe packaging cell line. Under these conditions the modified retroviralgenome including the inserted therapeutic and marker genes istranscribed from the vector plasmid and packaged into the modifiedretroviral particles (recombinant viral particles). This recombinantvirus is then used to infect target cells in which the vector genome andany carried marker or therapeutic genes becomes integrated into thetarget cell's DNA. A cell infected with such a recombinant viralparticle cannot produce new vector virus since no viral proteins arepresent in these cells. However, the DNA of the vector carrying thetherapeutic and marker genes is integrated in the cell's DNA and can nowbe expressed in the infected cell.

The recombinant MVA virus according to the invention expressing T7 RNApolymerase can be used to produce the proteins required for packagingretroviral vectors. To do this the gag, pol and env genes of aretrovirus (e.g. the Murine Leukemia Virus (MLV)) are placed undertranscriptional control of a T7 RNA polymerase promoter in one or moreexpression vectors (e.g. plasmids). The expression vectors are thenintroduced into cells infected with the recombinant MVA virus expressingT7 RNA polymerase, together with an expression vector carrying aretroviral vector construct, possibly under transcriptional control of aT7 RNA polymerase promoter.

WO 94/2943 7, WO 89/11539 and WO 96/7748 describes different types ofretroviral vector which can be packaged using the packaging systemdescribed above.

A further use of the recombinant MVA virus expressing T7 RNA polymeraseis to generate recombinant proteins, noninfectious virus particles, orinfectious mutant virus particles for the production of vaccines ortherapeutics (Buchholz et al., Virology, 204:770-776 (1994) andEP-B1-1356695)). To do this viral genes (e.g. the gag-poland env genesof HIV-1) are placed under transcriptional control of the T7 promoter inan expression vector (e.g. plasmid or another recombinant MVA virus).This construct is then introduced into cells infected with therecombinant MVA virus expressing T7 RNA polymerase. The recombinantviral genes are transcribed with high efficiency, recombinant proteinsare made in high amounts and can be purified. Additionally, expressedrecombinant viral proteins (e.g., HIV-1 env, gag) may assemble to viralpseudo-particles that budd from the cells and can be isolated from thetissue culture medium. In another embodiment, viral proteins (from e.g.HIV, SIV, Measles virus) expressed by the MVA-T7 pol system may rescuean additionally introduced mutant virus (derived from e.g. HIV, SIV,Measles virus) by overcoming a defect in attachment and infection,uncoating, nucleic acid replication, viral gene expression, assembly,budding or another step in viral multiplication to allow production andpurification of the mentioned mutant virus.

MVA-T7pol can also be used together with DNA sequences carrying the geneof an antigen of interest (e.g. the gene of HIV, nef, tat, gag, pol, orenv or others) for immunization. First, a coding sequence of a givenantigen (e.g HIV, HCV, HPV, HSV, measles virus, influenza virus orother) are cloned under control of a T7 RNA polymerase promoterpreferably in a plasmid vector and the resulting DNA construct isamplified and purified using standard laboratory procedures. Secondly,the vector DNA is inoculated simultaneously or with appropriate limelagstogether with MVA-T7pol. At the site of inoculation the recombinant geneof interest is expressed transiently in cells containing both the vectorDNA and MVA-T7 pol and the corresponding antigen is presented to thehost immune system stimulating an antigen-specific immune response. Thisprotocol using the non-replication vaccinia with MVA -T7 pol representsa promising novel approach to nucleic acid vaccination allowingefficient transient expression of a given antigen, but avoiding thepotential risk of constitutive gene expression.

The Recombinant MVA Vaccinia Viruses can be Prepared as Set OutHereinafter

A DNA-construct which contains a DNA-sequence which codes for a foreignpolypeptide flanked by MVA DNA sequences adjacent to a naturallyoccurring deletion, e.g. deletion II, within the MVA genome, isintroduced into cells infected with MVA, to allow homologousrecombination.

Once the DNA-construct has been introduced into the eukaryotic cell andthe foreign DNA has recombined with the viral DNA, it is possible toisolate the desired recombinant vaccinia virus in a manner known per se,preferably with the aid of a marker (compare Nakano et al., Proc. Natl.Acad. Sci. USA, 79:1593-1596 (1982); Franke et al., Mol. Cell. Biol,1918-1924 (1985); Chakrabarfi et al., Mol. Cell. Biol., 3403-3409(1985); Fathi et al., Virology 97-105 (1986)).

The DNA-construct to be inserted can be linear or circular. A circularDNA is preferred, especially a plasmid. The DNA-construct containssequences flanking the left and the right side of a naturally occurringdeletion, e.g. deletion II, within the MVA genome (Altenburger, W.,Suter, C. P. and Altenburger J., Arch. Virol., 105:15-27 (1989)). Theforeign DNA sequence is inserted between the sequences flanking thenaturally occurring deletion. The foreign DNA sequence can be a genecoding for a therapeutic polypeptide, e.g. t-PA or interferon, or anantigenic determinant from a pathogenic agent. Pathogenic agents can beviruses, bacteria and parasites which may cause a disease, as well astumor cells which multiply unrestrictedly in an organism and may thuslead to pathological growths. Examples of such pathogenic agents aredescribed in Davis, B. D. et al., (Microbiology, 3rd ed., Harperinternational Edition). Preferred antigens of pathogenic agents arethose of human immunodeficiency viruses (e.g. HIV-1 and HIV-2), ofmycobacteria causing tuberculosis, of the parasite PlasmodiumFalciparum, and of melanoma cells.

For the expression of a DNA sequence or gene, it is necessary forregulatory sequences, which are required for the transcription of thegene, to be present on the DNA. Such regulatory sequences (calledpromoters) are known to those skilled in the art, and includes forexample those of the vaccinia 11 kDa gene as are described inEP-A-198,328, and those of the 7.5 kDa gene (EP-A-110,385).

The DNA-construct can be introduced into the MVA infected cells bytransfection, for example by means of calcium phosphate precipitation(Graham et al., Virol., 52:456-467 (1973); Wigler et al., Cell 777-785(1979)) by means of electroporation (Neumann et al., EMBO J., 1:841-845(1982)), by microinjection (Graessmann et al., Meth. Enzymol.101:482-492 (1983)), by means of liposomes (Straubinger et al., Methodsin Enzymology, 101:512-527 (1983)), by means of spheroplasts (Schaffner,Proc. Natl. Acad. Sci. USA, 77:2163-2167 (1980)) or by other methodsknown to those skilled in the art. Transfection by means of calciumphosphate precipitation is preferred.

The detailed examples which follow are intended to contribute to abetter understanding of the present invention. However, it is notintended to give the impression that the invention is confined to thesubject-matter of the examples.

EXAMPLES

1. Growing and Purification of the Viruses

1.1 Growing of the MVA Virus

The MVA virus is a highly attenuated vaccinia virus derived from thevaccinia virus strain Ankara (CVA) by long-term serial passages onprimary chicken embryo fibroblast (CEF) cultures. For a general rewiewof the history of the production, the properties and the use of MVAstrain, reference may be made to the summary published by Mayr et al.,in Infection, 3:6-14 (1975). Due to the attenuation in CEF, the MVAvirus replicates to high titers in this avain host cell. In mammaliancells, however, MVA is severely growth restricted, and typical plaqueformation by the virus is not detectable. Therefore, MVA virus was grownon CEF cells. To prepare CEF cells, 11-day-old embryos were isolatedfrom incubated chicken eggs, the extremities are removed, and theembryos are minced and dissociated in a solution composed of 0.25%trypsin at 37° C. for 20 minutes. The resulting cell suspension wasfiltered and cells were sedimented by centrifugation at 2000 rpm in aSorvall RC-3B centrifuge at room temperature for 5 minutes, resuspendedin 10 volumes of medium A (MEM Eagle, for example obtainable from LifeTechnologies GmbH, Eggenstein, Germany), and sedimented again bycentrifugation at 2000 rpm in a Sorvall RC-3B centrifuge at roomtemperature for 5 minutes. The cell pellet was reconstituted in medium Acontaining 10% fetal calf serum (FCS), penicillin (100 units/mi),streptomycin (100 mg/ml) and 2 mM glutamine to obtain a cell suspensioncontaining 500,000 cells/ml. CEF cells obtained in this way were spreadon cell culture dishes. They were left to grow in medium A in a CO₂incubator at 37° C. for 1-2 days, depending on the desired cell density,and were used for infection either directly or after one further cellpassage. A detailed description of the preparation of primary culturescan be found in the book by R. I. Freshney, “Culture of animal cell,Alan R. Liss Verlag, New York (1983) Chapter 11, page 99 et seq.

MVA viruses were used for infection as follows. CEF cells were culturedin 175 cm² cell culture bottles. At 90-100% confluence, the medium wasremoved and the cells were incubated for one hour with an MVA virussuspension (0.01 infectious units (IU) per cell, 0.02 ml/cm²) in mediumA. Then more medium A was added (0.2 ml/cm²) and the bottles wereincubated at 37° C. for 2-3 days (until about 90% of the cells showcytopathogenic effect). Crude virus stocks were prepared by scrapingcell monolayers into the medium and pelleting the cell material bycentrifugation at 3000 rpm in a Sorvall RC-3B centrifuge at 4° C. for 5minutes. The crude virus preparation was stored at −20° C. beforeprocessing (e.g., virus purification).

1.2 Purification of the Viruses

The purification steps undertaken to obtain a virus preparation whichwas as pure as possible and free from components specific to the hostcell were similar to those described by Joklik, Virology, 18:9-18(1962)). Crude virus stocks which had been stored at −20° C. were thawedand suspended once in PBS (10-20 times the volume of the sediment), andthe suspension was centrifuged as above. The new sediment was suspendedin 10 times the volume of Tris buffer 1 (10 mM Tris-HCl pH 9.0,), andthe suspension was briefly treated with ultrasound (Labsonic L, B. BraunBiotech International, Melsungen Germany; 2×10 seconds at 60 watts androom temperature) in order to further disintegrate cell debris and toliberate the virus particles from the cellular material. The cell nucleiand the larger cell debris were removed in the subsequent briefcentrifugation of the suspension (Sorvall GSA rotor obtainable fromDuPont Co., D-6353 Bad Nauheim, FRG; 3 minutes at 3000 rpm and 10° C.).The sediment was once again suspended in Tris buffer 1, treated withultrasound and centrifuged, as described above. The collectedsupernatants containing the free virus particles were combined andlayered over a cushion of 10 ml of 36% sucrose in 10 mM Tris-HCl, pH9.0, and centrifuged in a Beckman SW 27/SW 28 rotor for 80 minutes with13,500 rpm at 40° C. The supernatant was discarded, and the sedimentcontaining the virus particles was taken up in 10 ml of 1 mM Tris-HCl,pH 9.0, homogenized by brief treatment with ultrasound (2×10 seconds atroom temperature, apparatus as described above), and applied to a 20-40%sucrose gradient (sucrose in 1 mM Tris-HCl, pH 9.0) for furtherpurification. The gradient was centrifuged in Beckmann SW41 rotor at13,000 rpm for 50 minutes at 4° C. After centrifugation, discrete bandscontaining virus particles were harvested by pipetting after aspiratingvolume above band. The obtained sucrose solution was diluted with threevolumes PBS and the virus particles were sedimented again bycentrifugation (Beckmann SW 27/28, 60 minutes at 13,500 rpm, 4° C.). Thepellet, which now consisted mostly of pure virus particles, wasresuspended in PBS and equilibrated to virus concentrationscorresponding on average to 1-5×10⁹ IU/ml. The purified virus stocksolution was stored at −80° C. and used either directly or diluted withPBS for subsequent experiments.

1.3 Cloning of MVA Virus

To generate homogeneous stock virus preparations MVA virus obtained fromProf. Anton Mayr was cloned by limiting dilution during threeconsecutive passages in CEF cultured on 96-well tissue culture plates.The MVA clone F6 was selected and amplified in CEF to obtain workingstocks of virus that served as starting material for the generation ofrecombinant MVA viruses described in this patent application as well asfor the generation of recombinant MVA viruses described previously(Sutter, G. and Moss, B., Proc. Natl. Acad. Sci. USA, 89:10847-10851(1992); Sutter, G. et al., Vaccine, 12:1032-1040 (1994); Hirsch, V. etal., J. Virol., 70:3741-3752 (1996)).

2. Construction and Characterization of Recombinant MVA Viruses

2.1. Construction of Vector Plasmids

To allow the generation of recombinant MVA viruses novel vector plasmidswere constructed. Insertion of foreign genes into the MVA genome wastargeted precisely to the site of the naturally occurring deletion II inthe MVA genome. Sequences of MVA DNA flanking the site of a 2500-bpdeletion in the HindIII N fragment of the MVA genome (Altenburger, W. etal., J. Arch. Virol., 105:15-27 (1989)) were amplified by PCR and clonedinto the multiple cloning site of plasmid pUC18. The primers for theleft 600-bp DNA flank were 5′-CAG CAG GGT ACC CTC ATC GTA CAG GAC GTTCTC-3′ (SEQ ID NO: 1) and 5′-CAG CAG CCC GGG TAT TCG ATG ATT ATT TTT AACAAA ATA ACA-3′ (SEQ ID NO: 2) (sites for restriction enzymes Kpnl andSmal are underlined).

The primers for the right 550-bp DNA flank were 5′-CAG CAG CTG CAG GAATCA TCC ATT CCA CTG AAT AGC-3′ (SEQ ID NO: 3); and 5′-CAG CAG GCA TGCCGA CGA ACA AGG AAC TGT AGC AGA-3′ (SEQ ID NO: 4)(sites for restrictionenzymes Pstl and Sphl are underlined). Between these flanks of MVA DNAinserted in pUC18, the Escherichia coli LacZ gene under control of thevaccinia virus late promoter P11 (prepared by restriction digest frompIII LZ, Sutter, G. and Moss, B., PNAS USA 89:10847-10851(1992)) wasinserted, using the BamHI site, to generate the MVA insertion vectorpUCII LZ (FIG. 1). In the following, a 289 bp fragment containing thevaccinia virus early-late promoter P7.5 together with a Smal site forcloning (prepared by restriction digest with EcoRI and Xbal from theplasmid vector pSC11 (Chakrabarbti et al., Mole. Cell. Biol.,5:3403-3409 (1985)) was inserted into the Smal site of pUCII LZ to givethe MVA vector pUC II LZ P7.5 [FIG. 2]. To construct a vector plasmidthat allows isolation of recombinant MVA viruses via transient synthesisof the reporter enzyme β-galactosidase a 330 bp DNA fragment from the3′-end of the E. coli LacZ open reading frame was amplified by PCR(primers were 5′-CAG CAG GTC GAC CCC GAC CGC CTT ACT GCC GCC-3′ (SEQ IDNO: 5) and 5′-GGG GGG CTG CAG ATG GTA GCG ACC GGC GCT CAG-3′ (SEQ ID NO:6)) and cloned into the SalL and Pstl sites of pUC II LZ P7.5 to obtainthe MVA vector pUC II LZdel P7.5 (FIG. 3). Using the Smal site, thisvector plasmid can be used to insert DNA sequences encoding a foreigngene under transcriptional control of the vaccinia virus promoter P7.5into the MVA genome. After the desired recombinant virus has beenisolated by screening for expression of β-galactosidase activity furtherpropagation of the recombinant virus leads to the self-deletion of thereengineered P11-LacZ expression cassette by homologous recombination.

2.2. Construction and Characterization of Recombinant Virus MVA T7pol

A 3.1 kbp DNA fragment containing the entire gene of bacteriophage T7RNA polymerase under control of the vaccinia virus early/late promoterP7.5 was excised with EcoRl from plasmid pTF7-3 (Fuerst, T. R. et al.,P.N.A.S. USA, 83:8122-8126 (1986), modified by incubation with KlenowDNA polymerase to generate blunt ends, and cloned into a unique Smalrestriction site of pUCII LZ to make the plasmid transfer vector pUCIILZ T7pol (FIG. 4). As transcriptional regulator for the expression ofthe T7 RNA polymerase gene the vaccinia virus early/late promoter P7.5was chosen. Contrary to stronger vaccinia virus late promoters (e.g.P11) this promoter system allows expression of recombinant genesimmediately after the infection of target calls. The plasmid pUCII LZT7pol that directs the insertion of the foreign-genes into the site ofdeletion II of the MVA genome was used to generate the recombinant virusMVA T7pol.

CEF cells infected with MVA at a multiplicity of 0.05 TCID₅₀ per cellwere transfected with DNA of plasmid pUCII LZ T7pol as describedpreviously (Sutter, G, et al., Vaccine, 12:1032-1040 (1994)).Recombinant MVA virus expressing the T7 RNA polymerase and co-expressingβ-D-galactosidase (MVA P7.5-T7pol) was selected by five consecutiverounds of plaque purification in CEF cells stained with5-bromo-4-chloro-3-indolyl β-D-galactoside (300 μg/ml). Subsequently,recombinant viruses were amplified by infection of CEF monolayers, andthe DNA was analyzed by PCR to confirm genetic homogeneity of the virusstock. Southern blot analysis of MVA-T7pol viral DNA demonstrated stableintegration of the recombinant genes at the site of deletion II withinthe MVA genome.

To monitor expression of T7 RNA polymerase by recombinant MVA T7pol[³⁵S]methionine -labeled polypeptides from virus infected tissue culturewere analyzed. Monolayers of the monkey kidney cell line CV-1 grown in12-well plates were infected with virus at a multiplicity of 20 TCID₅₀per cell. At 3 to 5 hours after infection, the medium was removed, andthe cultures were washed once with 1 ml of methionine free medium. Toeach well, 0.2 ml of methionine-free medium supplemented with 50 μCi of[³⁵S]methionine was added and incubated for 30 minutes at 37° C.Cytoplasmic extracts of infected cells were prepared by incubating eachwell in 0.2 ml of 0.5% Nonidet P-40 lysis buffer for 10 mm at 37° C. andsamples were analyzed by SDS-PAGE. The metabolic labeling of the CV-1cells with MVA T7pol revealed the synthesis of two additionalpolypeptides (i) a protein of about 116,000 Da representing the E. coliβ-galactosidase co-expressed to allow the screening for recombinantvirus and (ii) a 98,000 Da protein with the expected size of thebacteriophage T7 RNA polymerase. The large amount of β-galactocidasemade by MVA T7pol is remarkable. The results from the in vivo labelingexperiments demonstrate a very strong expression of the P11-LacZ geneconstruct when inserted into the MVA genome at the site of deletion IIindicating that recombinant genes in MVA vector viruses might beexpressed more efficiently when inserted into this locus of the MVAgenome.

The usefulness of MVA-T7pol recombinant viruses as expression system incomparison to the WR-T7pol recombinant virus vTF7-3 (Fuerst et al. 1986)was tested by the co-transfection of DNA of a plasmid vector that isderived from pTMl (Moss, B., et al., Nature, 348:91-92 (1990)) andcontains (cloned into the Ncol and BamHI sites of the pTM 1 multiplecloning site) the E. coli chloramphenicol acetyltranferase (CAT) geneunder the control of a T7 RNA polymerase promoter (PT₇). Transfected andinfected CV-1 cells were suspended in 0.2 ml of 0.25 M Tris-HCl (pH7.5). After three freeze-thaw cycles, the lysates were cleared bycentrifugation, the protein content of the supernatants was determined,and samples containing 0.5, 0.25, 0.1 μg total protein were assayed forenzyme activity as described by Mackett, M., et al., J. Virol.,49:857-864 (1984). After autoradiography, labeled spots were quantitatedusing the Fuji imaging analysis system.

The results demonstrate that by using the highly attenuated vacciniavector MVA it is possible to exploit the vaccinia virus-T7 RNApolymerase system as efficiently as by using a fullyreplication-competent vaccinia virus recombinant.

2.3. Construction and Characterization of Recombinant Virus MVA-LAInef

A 648 bp DNA fragment containing the entire nef gene of HIV-1 LAI wasprepared by PCR from plasmid DNA (pTG1166 kindly provided by M.-P.Kieny, Transgene S.A., Strasbourg; PCR primers were 5′-CAG CAG GGA TCCATG GGT GGC AAG TGG TCA AAA AGT AGT-3′ (SEQ ID NO: 7) and 5′-CAG CAG GGATCC ATG TCA GCA GTT CTT GAA GTA CTC CGG-3′ (SEQ ID NO: 8)), digestedwith restriction endonuclease BamHI, modified by incubation with KlenowDNA polymerase to generate blunt ends, and cloned into the Smal site ofpUC II LZdel P7.5 to make the vector pUC II LZdel P7.5-LAInef (FIG. 5).This plasmid could be used to engineer MVA recombinant virus thatexpresses the nef gene of HIV-1 LAI under control of the vaccinia virusearly/late promoter P7.5.

CEF cells infected with MVA at a multiplicity of 0.05 TCID₅₀ per cellwere transfected with DNA of plasmid pUC II LZdel P7.5-LAInef asdescribed previously (Sutter, G. et al., Vaccine, 12:1032-1040 (1994)).Recombinant MVA viruses containing the nefgene and transientlyco-expressing the E. coli LacZ marker gene were selected by consecutiverounds of plaque purification in CEF cells stained with5-bromo-4-chloro-3-indolyl β-D-galactoside (300 μg/ml). In thefollowing, recombinant MVA viruses containing the nef gene and havingdeleted the LacZ marker gene were isolated by three additionalconsecutive rounds of plaque purification screening for non-stainingviral foci in CEF cells in the presence of 5-bromo-4-chloro-3-indolylβ-galactoside (300 μg/ml). Subsequently, recombinant viruses wereamplified by infection of CEF monolayers, and the MVA-LAInef viral DNAwas analyzed by PCR to confirm genetic homogeneity of the virus stock.Southern blot of viral DNA confirmed genetic stability of MVA-LAlnef andprecisely demonstrated integration of the nef gene and deletion of theE. coli LacZ marker gene at the site of deletion II within the viralgenome.

Efficient expression of recombinant Nef protein was confirmed by Westernblot analysis of protein lysates from CEF cells infected with MVA-LAInefusing mouse monoclonal antibodies directed against HIV-1 Nef (kindlyprovided by K. Krohn and used as described by Ovod, V. et al., AIDS,6:25-34 (1992)).

2.4. Construction and Characterization of Recombinant Virus MVA-hTYR

A 1.9 kb DNA fragment containing the entire gene encoding humantyrosinase (Tyrosinase c-DNA clone 123.B2 isolated from the melanomecell line SK29-MEL of patient SK29 (AV), GenBank Acct. No. U01873;Brichard, V. et al., J. Exp. Med., 178:489-495 (1993)) was prepared fromthe plasmid pcDNAI/Amp-Tyr (Wolfel, T. et al., Eur. J. Immunol.,24:759-764 (1994)) by EcoRI digest, modified by incubation with KlenowDNA polymerase to generate blunt ends, and cloned into the Smal site ofpUC II LZdel P7.5 to make the vector pUC II LZdel P7.5-TYR (FIG. 6).

This plasmid could be used to engineer MVA recombinant virus thatexpresses the human tyrosinase gene under control of the vaccinia virusearly/late promoter P7.5.

CEF cells infected with MVA at a multiplicity of 0.05 TCID₅₀ per cellwere transfected with DNA of plasmid pUC II LZdel P7.5-TYR as describedpreviously (Sutter, G, et al., Vaccine, 12:1032-1040 (1994)).Recombinant MVA virus stably expressing the gene for human tyrosinaseand transiently co-expressing the E. coli LacZ gene was selected byconsecutive rounds of plaque purification in CEF cells stained with5-bromo-4-chloro-3-indolyl β-D-galactoside (300 μg/ml). In thefollowing, recombinant MVA virus expressing the gene encoding humantyrosinase and having deleted the LacZ marker gene was isolated by threeadditional consecutive rounds of plaque purification screening fornon-staining viral foci in CEF cells in the presence of5-bromo-4-chloro-3-indolyl μ-D-galactoside (300 μg/ml). Subsequently,recombinant viruses were amplified by infection of CEF monolayers, andthe MVA-hTYR viral DNA was analyzed by PCR to confirm genetichomogeneity of the virus stock. Southern blot analysis of viral DNAconfirmed genetic stability of MVA-hTYR and precisely demonstratedintegration of the recombinant tyrosinase gene and deletion of the E.coli LacZ marker gene at the site of deletion II within the viralgenome.

Efficient expression of recombinant human tryosinase was confirmed byWestern blot analysis of protein lysates from CEF cells infected withMVA-hTYR using rabbit polyclonal antibodies (kindly provided by V.Hearing and used as described by Jimenez, M., et al., P.NA.S. USA,85:3830-3834 (1988)) or mouse monoclonal antibodies (kindly provided byL. Old and used as described by Chen, Y. et al., P.N.A.S. USA92:8125-8129 (1995)) directed against tyrosinase.

Equivalents

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims. Those skilled in the artwill recognize or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described specifically herein. Such equivalents are intendedto be encompassed in the scope of the claims.

1-30. (canceled)
 31. A method of inducing an immune response againstHIV-1 nef in an HIV-1 infected host comprising: administering to theHIV-1 infected host a Modified Vaccinia Virus Ankara (MVA) containing aforeign gene encoding HIV-1 nef, wherein, upon administration to theHIV-1 infected host, the HIV-1 nef in the recombinant MVA is expressedand induces an immune response.
 32. The method of claim 31, wherein theforeign gene is inserted at a site of a naturally occurring deletionwithin an MVA genome selected from the group consisting of deletion siteI, site II, site IV, site V, and site VI, and site VI.
 33. The method ofclaim 32, wherein the site of a naturally occurring deletion within anMVA genome is deletion site I.
 34. The method of claim 32, wherein thesite of a naturally occurring deletion within an MVA genome is deletionsite II.
 35. The method of claim 32, wherein the site of a naturallyoccurring deletion within an MVA genome is deletion site IV.
 36. Themethod of claim 32, wherein the site of a naturally occurring deletionwithin an MVA genome is deletion site V.
 37. The method of claim 32,wherein the site of a naturally occurring deletion within an MVA genomeis deletion site VI.
 38. The method of claim 31, wherein the host is ahuman.
 39. The method of claim 31, wherein the foreign gene is under thecontrol of a T7 RNA polymerase promoter.
 40. The method of claim 31,wherein the foreign gene is under the control of the vaccinia virusearly/late promoter p7.5.